PROJECT SUMMARY/ABSTRACT Recovery of neuronal function is critical for overcoming botulinum neurotoxin (BoNT)-mediated paralysis. Strategies for promoting such recovery have proven nearly intractable. The proposed research seeks to identify novel small molecules that act directly on neuronal processes/functions to mitigate BoNT intoxication, regardless of the serotype, by means other than inhibition of the toxins' enzymatic activities. BoNTs are the most potent of the biological toxins and may be delivered via food spiking and/or aerosol route. Currently, long-term mechanical ventilation is the only life-sustaining option once the BoNT-mediated paralysis of diaphragm muscles manifests. Consequently, there is a significant void that needs to be filled: the discovery and development of therapeutics that will counter BoNTs post-exposure and/or expedite neuronal recovery. Nearly all drug development efforts focus solely on inhibiting the Zn metalloprotease light chain (LC) components of these toxins; BoNT/LCs selectively cleave different SNARE complex subunits involved in neurotransmitter release. Seven biochemically distinct BoNT serotypes (A-G) cause botulism, making the prospect of developing a single, non-promiscuous inhibitor of all serotypes unlikely. We believe targeting BoNT/LCs must be accompanied by other endeavors to enhance neuronal recovery. Hence, broadening the scope of viable targets to include cellular processes involved in either intoxication or recovery may provide novel methods for countering multiple serotypes. However, this strategy has been hampered by a lack of high- throughput cell-based assays that measure BoNT activity. Our group developed a comprehensive system consisting of human or mouse motor neurons derived from embryonic stem cells, and imaging assays using BoNT cleavage-sensitive antibodies. Thus, for the first time, a system is available to identify small molecules that can impinge on neuronal pathways in addition to the toxin itself. We propose taking advantage of our unique capabilities to identify pharmacologically active small molecules with unique mechanisms of action for countering BoNT/A, /B and /E poisoning. Following identification, lead compounds will be characterized and optimized via rounds of chemical synthesis and biological evaluation. Optimized compounds will be evaluated in animal models of intoxication to identify viable candidates for advanced development as therapeutic countermeasures. To guide our studies, we offer the following hypothesis: It is possible to counter the effects of multiple BoNTs by small molecules which act on neuronal cell functions by a means other than blockade of the enzymatic site of the toxin. The main goal of this proposal is to identify compounds that are effective against BoNTs A, B and E post- intoxication to serve as chemical leads for advanced development studies. A second goal is to use the identified lead compounds as chemical probes for dissecting the neuronal signaling pathways that are required by these toxins. To accomplish these goals, we will conduct the first ever large-scale cell-based screening to identify BoNT/A, /B, and /E inhibitors and optimize lead molecules using pharmacophore-based approaches incorporating medicinal chemistry, organic synthesis, in vitro and in vivo testing, and molecular modeling.